Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.
The overall goals of this procedure are to assess how mitochondrial function alterations affect the development of neurological disorders and to design therapeutics that have a direct effect on blood-brain barrier openings. This method can help answer key questions in the field of neurological disorders, such as how can neuronal cell death be minimized following a stroke? The main advantage of this technique is that it provides a real-time assessment of mitochondrial function in whole intact cells.
After growing the cerebral vascular endothelial cells in complete medium, in a T75 centimeter squared tissue culture flask, at 37 degrees Celsius and 5%carbon dioxide to 80%confluency, discard the cell culture medium, and rinse the cells with trypsin EDTA. Then add one to two milliliters of fresh trypsin EDTA to the flask and detach the cells at 37 degrees Celsius for three to five minutes. After confirming the cells have lifted under an inverted microscope, stop the reaction with gentle addition of 10 milliliters of fresh complete cell medium and transfer the cells to a 15 milliliter conical tube for centrifugation.
Discard the supernatant and wash the pellet in 10 milliliters of fresh complete growth medium for another centrifugation. Then re-suspend the pellet in complete growth medium at a two times 10 to the fifth cells per milliliter concentration for subculture. After the appropriate number of subcultures, seed 1.6 times 10 to the fourth cells in 80 microliters of complete medium per well, onto a 96-well extracellular flux cell culture plate and place the plate in a 37 degree Celsius incubator with 5%carbon dioxide.
When the cells have attached to the plate, add the treatment of interest to the appropriate wells and return the cells to the incubator. The day before the mitochondrial functional assessment assay, hydrate an extracellular flux censor cartridge with 150 microliters of extracellular flux calibrant solution and incubate the plate over night at 37 degrees Celsius without carbon dioxide. The next morning, use the plate washer station to change the cell culture medium into pH 7.0 extracellular flux assay medium.
Then, incubate the cells at 37 degrees Celsius, without carbon dioxide, for 30 to 60 minutes. When the cells have equilibrated, load the hydrated cartridge into the Bioanalyzer and click the Start button to begin the calibration. At the end of the calibration, click the Unlock Cartridge prompt to remove the cartridge bottom plate and load the cell plate.
Then open the data file to obtain the rate values from the Bioanalyzer for export into the spreadsheet file. Given that the kinetics and relative intensity of the responses are varied among the different cerebral vascular endothelial cell types, in these experiments, a series of concentrations of bEnd. 3 cells was used to identify the optimal number of cells for measuring the oxygen consumption rate levels in a metabolic profiling assay.
As observed, the basal and maximal respirations and spare respiratory capacity demonstrate a proportional response to the cell density. ATP production, however, decreases when the highest concentration of cells per well is used, suggesting that an over confluent cell culture is not suitable for the experiment. With the optimal cell densities appearing to occur in the mid range of cell numbers per well.
Therefore, the expected oxygen consumption rate levels were assessed in this representative experiment using a mid range cell concentration, demonstrating that non coding precursor mRNA molecules reduce the mitochondrial function in cerebral vascular endothelial cells. These experiments also demonstrate that the maximal respiration and spare capacity are significantly decreased by the over expression of these molecules in cerebral vascular endothelial cells at 24 hours post transfection, even though the basal respiration, ATP production, and proton leak exhibit no significant changes. Once mastered, this technique can be completed in two days.
From plating the cells to accessing mitochondrial function, if it is performed properly. While attempting this procedure, it's important to remember that determining the optimal cell density is extremely important before beginning any other procedures. Following this procedure, other methods, like cell viability or glycolytic assays, can be performed to answer additional questions about the changes that occur in mitochondrial function due to a decreased cell viability or changes in other metabolic pathways.
After its development, this technique paved the way for researchers in the field of mitochondrial function for using intact cells inside of isolated mitochondria and a variety of diseases, including neurological diseases, cancer, or metabolic disorders. After watching this video, you should have a good understanding of how to access mitochondrial function in cerebral vascular endothelial cells.
